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  • 1.
    Ahrenstedt, Lage
    et al.
    KTH, Skolan för bioteknologi (BIO). KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Olksanen, Antti
    VTT Technical Research Centre of Finland.
    Salmien, Kristian
    VTT Technical Research Centre of Finland.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Paper dry strength improvement by xyloglucan addition: Wet-end application, spray coating and synergism with borate2008Ingår i: Holzforschung, ISSN 0018-3830, E-ISSN 1437-434X, Vol. 62, nr 1, s. 8-14Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The polysaccharide xyloglucan as a wet-end additive improves paper properties. In the present study, paper strength improvement was analysed for dry handsheets made from chemical, mechanical and recycled pulps coated with xyloglucan in a spray application. Results are compared with sheets made from the same pulps treated with xyloglucan in the wet-end. Kraft pulp handsheets of bleached hardwood and softwood showed significant improvements of tensile, tear and Z-strength by xyloglucan spray treatment versus wet-end application, whereas handsheets of de-inked and thermomechanical pulp were improved only slightly. In both wet-end and spray applications, the effect of xyloglucan addition was intimately related to the presence of non-cellulosic components on the fibre surface. Further strength improvements were obtained for chemical pulps by addition of borax to the spray solution, which were likely to be due to the formation of borate-mediated xyloglucan cross-links. Spray coating of xyloglucan, with or without borax, thus represents a potential new application of this polysaccharide to increase paper dry strength.

  • 2. Anasontzis, George E.
    et al.
    Pena, Margarita Salazar
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Olsson, Lisbeth
    Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris2014Ingår i: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 30, nr 3, s. 728-735Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed.

  • 3.
    Araújo, Ana Catarina
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Nakhai, Azadeh
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ruda, M.
    Slättegård, R.
    Gatenholm, P.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    A general route to xyloglucan-peptide conjugates for the activation of cellulose surfaces2012Ingår i: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 354, s. 116-120Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellulose is an attractive supporting matrix for diverse biotechnological applications, including chromatography, diagnostics, and tissue replacement/scaffolding, due to its renewable resource status, low cost, and low non-specific interaction with biomolecules. In an effort to expand the biofunctionality of cellulose materials, we present here a versatile method for the synthesis of xyloglucan-peptide conjugates that harness the strong xyloglucan-cellulose binding interaction for gentle surface modification. Xylogluco-oligosaccharide aminoalditols (XGO-NH 2) were coupled to both linear and cyclic peptides, which contained the endothelial cell epitope Arg-Gly-Asp, in a facile two-step approach employing diethyl squarate cross-linking. Subsequent xyloglucan endo-transglycosylase-mediated coupling of the resulting XGO-GRGDS (Gly-Arg-Gly-Asp-Ser) and XGO-c[RGDfK]-PEG-PEG (cyclo[Arg-Gly-Asp-(d-Phe)-Lys]-PEG-PEG; where PEG is 8-amino-3,6-dioxaoctanoic acid) conjugates into high molecular mass xyloglucan yielded xyloglucan-RGD peptide conjugates suitable for cellulose surface activation. Notably, use of XGO-squaramate as a readily accessible, versatile intermediate overcomes previous limitations of solid-phase synthetic approaches to XGO-peptide conjugates, and furthermore allows the method to be generalized to a wide variety of polypeptides and proteins, as well as diverse primary amino compounds.

  • 4.
    Araújo, Ana Catarina
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Song, Yajing
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ståhl, Patrik L.
    Brumer, Harry, III
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport2012Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, nr 7, s. 3311-3317Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.

  • 5. Ariza, A.
    et al.
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Offen, W.A.
    Roberts, S.M.
    Wilson, K.S.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Davies, G.J.
    Structure and Activity of a Paenibacillus polymyxa Xyloglucanase from Glycoside Hydrolase Family 442011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 39, s. 33890-33900Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the "accessory enzymes," including xyloglucanases, that may aid biomass degradation through removal of "hemicellulose" polysaccharides. Here, we report the biochemical characterization of the endo-beta-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the -4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the -3 to -1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the -4' subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

  • 6.
    Aspeborg, Henrik
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Coutinho, Pedro M.
    Wang, Yang
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Henrissat, Bernard
    Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)2012Ingår i: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 12, nr 1, s. 186-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on beta-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results: About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion: Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

  • 7.
    Baumann, Martin J.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Eklöf, Jens M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Michel, Gurvan
    Kallas, Åsa M.
    KTH, Skolan för bioteknologi (BIO).
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Czjzek, Mirjam
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: Biological implications for cell wall metabolism2007Ingår i: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 19, nr 6, s. 1947-1963Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen ( Populus tremula 3 Populus tremuloides). Production of the loop deletion variant Tm-NXG1-Delta YNIIG yielded an enzyme that was structurally similar to Ptt- XET16-34 and had a greatly increased transglycosylation: hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.

  • 8.
    Baumann, Martin J.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO).
    Michel, G.
    Kallas, Åsa
    KTH.
    Teeri, Tuula
    KTH, Skolan för bioteknologi (BIO).
    Czjzek, Mirjam
    KTH.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structural analysis of nasturtium NXG reveals the evolution of GH16 xyloglucanase activity from XETs: biological implications for cell wall metabolismManuskript (Övrigt vetenskapligt)
  • 9. Becker, D.
    et al.
    Braet, C.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Claeyssens, M.
    Divne, Christina
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Fagerstrom, B. R.
    Harris, M.
    Jones, T. A.
    Kleywegt, G. J.
    Koivula, A.
    Mahdi, S.
    Piens, K.
    Sinnott, M. L.
    Stahlberg, J.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Underwood, M.
    Wohlfahrt, G.
    Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant2001Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 356, s. 19-30Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

  • 10.
    Benselfelt, Tobias
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Fiberteknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Cranston, Emily D.
    Department of Chemical Engineering, McMaster University.
    Ondaral, Sedat
    Department of Pulp and Paper Technology, Karadeniz Technical University.
    Johansson, Erik
    Cellutech AB.
    Brumer, Harry
    The Michael Smith Laboratories and the Department of Chemistry, The University of British Columbia.
    Rutland, Mark W.
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Wågberg, Lars
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Fiberteknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Adsorption of Xyloglucan onto Cellulose Surfaces of Different Morphologies: An Entropy-Driven Process2016Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 17, nr 9, s. 2801-2811Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The temperature-dependence of xyloglucan (XG) adsorption onto smooth cellulose model films regenerated from N-methylmorpholine N-oxide (NMMO) was investigated using surface plasmon resonance spectroscopy, and it was found that the adsorbed amount increased with increasing temperature. This implies that the adsorption of XG to NMMO-regenerated cellulose is endothermic and supports the hypothesis that the adsorption of XG onto cellulose is an entropy-driven process. We suggest that XG adsorption is mainly driven by the release of water molecules from the highly hydrated cellulose surfaces and from the XG molecules, rather than through hydrogen bonding and van der Waals forces as previously suggested. To test this hypothesis, the adsorption of XG onto cellulose was studied using cellulose films with different morphologies prepared from cellulose nanocrystals (CNC), semicrystalline NMMO-regenerated cellulose, and amorphous cellulose regenerated from lithium chloride/dimethylacetamide. The total amount of high molecular weight xyloglucan (XGHMW) adsorbed was studied by quartz crystal microbalance and reflectometry measurements, and it was found that the adsorption was greatest on the amorphous cellulose followed by the CNC and NMMO-regenerated cellulose films. There was a significant correlation between the cellulose dry film thickness and the adsorbed XG amount, indicating that XG penetrated into the films. There was also a correlation between the swelling of the films and the adsorbed amounts and conformation of XG, which further strengthened the conclusion that the water content and the subsequent release of the water upon adsorption are important components of the adsorption process.

  • 11.
    Bi, Ran
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Träkemi och massateknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi, Träkemi och massateknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Isolation and identification of microorganisms from soil able to live on lignin as acarbon source and to produce enzymes which cleave the β-o-4 bond in a lignin model compound2012Ingår i: Cellulose Chemistry and Technology, ISSN 0576-9787, Vol. 46, nr 3-4, s. 227-242Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several strains of fungi were isolated and identified from Scandinavian soil using agar plates with lignin as a carbon source. The strains grew significantly faster on this medium than on control plates without lignin. Different types of technical lignins were used, some of which contained trace amounts of sugars, even if the increased growth rate seemed not related to the sugar content. Some strains were cultivated in shaking flask cultures with lignin as a carbon source, with lignin apparently consumed by microbes - while accumulation of the microorganism biomass occurred. The cell-free filtrates of these cultures could reduce the apparent molecular weights of lignosulphonates, while the culture filtrate of one strain could cleave the beta-O-4 bond in a lignin model compound.

  • 12.
    Bi, Ran
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Lawoko, Martin
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Isolation and identification of microorganisms from soil able to live on lignin as a carbon source and to produce enzymes which cleave beta-O-4 bond in a lignin model compound2012Ingår i: Cellulose Chemistry and Technology, ISSN 0576-9787, Vol. 46, nr 3-4, s. 227-242Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several strains of fungi were isolated and identified from Scandinavian soil using agar plates with lignin as a carbon source. The strains grew significantly faster on this medium than on control plates without lignin. Different types of technical lignins were used, some of which contained trace amounts of sugars, even if the increased growth rate seemed not related to the sugar content. Some strains were cultivated in shaking flask cultures with lignin as a carbon source, with lignin apparently consumed by microbes - while accumulation of the microorganism biomass occurred. The cell-free filtrates of these cultures could reduce the apparent molecular weights of lignosulphonates, while the culture filtrate of one strain could cleave the beta-O-4 bond in a lignin model compound.

  • 13.
    Bi, Ran
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Spadiut, Oliver
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Lawoko, Martin
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Henriksson, Gunnar
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik.
    Isolation and identification of microorganisms from soil able to utilize lignin as single carbon source2011Ingår i: Proceedings of the 16th International Symposium of wood, fiber and pulp chemistry, 2011, s. 1091-1095Konferensbidrag (Refereegranskat)
  • 14.
    Bi, Ran
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Spaduit, Oliver
    KTH, Skolan för bioteknologi (BIO). KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Brumer, Harry III
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Isolation and identification of microorganisms from soil  able to ive on lignin as carbon source and produce enzymes that cleave beta-O-4mbond in lignin2011Ingår i: Cellulose Chemistry and Technology, ISSN 0576-9787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Twenty one strains of micro organism from Scandinavian soil had been isolated that could utilize lignin as only carbon source and 11 strains of them were identified. Different types of technical lignins were used.5 faster growing strains were cultivated in shaking flask cultures with ligninosulfonate as sole carbon source,and lignin appeared to be consumed after several days while mycelia was observed accumulated.Cell free filtrates of the 5 faster growing strains could lower the apparent molecular weights of lignosulphonates and the culture filtrate of one strain could cleave the lignin model compound with.The significances of the results are discussed.

  • 15.
    Bodin, Aase
    et al.
    Chalmers Tekniska Högskola.
    Ahrenstedt, Lage
    KTH, Skolan för bioteknologi (BIO).
    Fink, Helen
    Vascular Engineering Centre, Sahlgrenska University Hospital.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    Risberg, Bo
    Vascular Engineering Centre, Sahlgrenska University Hospital.
    Gatenholm, Paul
    Chalmers Tekniska Högskola.
    Modification of nanocellulose with a xyloglucan-RGD conjugate enhances adhesion and proliferation of endothelial cells: Implications for tissue engineering2007Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, nr 12, s. 3697-3704Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This paper describes a novel method for introducing the RGD cell adhesion peptide to enhance cell adhesion onto bacterial cellulose (BC). BC and cotton linters as reference were modified with xyloglucan (XG) and xyloglugan bearing a GRGDS pentapeptide. The adsorptions followed Langmuir adsorption behavior, where both XGs probably decorate the cellulose surfaces as a monolayer. The adsorption maximum of the XGs reached around 180 mg/g on BC and only about three times as much on cotton linters. The adsorption was verified with colorimetric methods. The specific surface area of BC measured with XG and XG-GRGDS was about 200 m(2)/g and was almost three times less for cotton linters, 60 m2/g. The difference in the amounts of XGs adsorbed might be explained by the swollen network of bacterial cellulose and a more exposed and accessible bulk as compared to cotton linters. The nanocellulose material was modified homogeneously throughout the material, as seen by the z-scan in confocal microscopy. Moreover, the modification in the water phase, in comparison with organic solvents, was clearly advantageous for preserving the morphology, as observed with SEM. The modification slightly increased the wettability, which might explain the decrease in or undetectable adsorption of adhesive protein shown by QCM-D. Initial cell studies showed that adhesion of human endothelial cells is enhanced when the BC hydrogel is modified with XG-GRGDS. QCM-D studies further revealed that the cell enhancement is due to the presence of the RGD epitope on XG and not to a nonspecific adsorption of fibronectin from cell culture medium. Optimization and proliferation studies of human endothelial cells onto bacterial cellulose modified with XG-GRGDS are currently being carried out at the Vascular Engineering Center, Sahlgrenska University Hospital, Gothenburg.

  • 16. Borriss, R.
    et al.
    Krah, M.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Kerzhner, M. A.
    Ivanen, D. R.
    Eneyskaya, E. V.
    Elyakova, L. A.
    Shishlyannikov, S. M.
    Shabalin, K. A.
    Neustroev, K. N.
    Enzymatic synthesis of 4-methylumbelliferyl (1 -> 3)-beta-D-glucooligosaccharides - new substrates for beta-1,3-1,4-D-glucanase2003Ingår i: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 338, nr 14, s. 1455-1467Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1 --> 3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1 --> 3)](n)-beta-D-Glcp-MeUmb, where n = 1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1 --> 3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1 --> 3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by H-1 and C-13 NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p. > 3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUrnbGlcP and MeUmbG(2).

  • 17. Bourquin, V.
    et al.
    Nishikubo, N.
    Abe, H.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Denman, S.
    Eklund, M.
    Christiernin, M.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Sundberg, B.
    Mellerowicz, E. J.
    Xyloglucan endotransglycosylases have a function during the formation of secondary cell walls of vascular tissues2002Ingår i: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 14, nr 12, s. 3073-3088Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Xyloglucan transglycosylases (XETs) have been implicated in many aspects of cell wall biosynthesis, but their function in vascular tissues, in general, and in the formation of secondary walls, in particular, is less well understood. Using an in situ XET activity assay in poplar stems, we have demonstrated XET activity in xylem and phloem fibers at the stage of secondary wall formation. Immunolocalization of fucosylated xylogucan with CCRC-M1 antibodies showed that levels of this species increased at the border between the primary and secondary wall layers at the time of secondary wall deposition. Furthermore, one of the most abundant XET isoforms in secondary vascular tissues (PttXET16A) was cloned and immunolocalized to fibers at the stage of secondary wall formation. Together, these data strongly suggest that XET has a previously unreported role in restructuring primary walls at the time when secondary wall layers are deposited, probably creating and reinforcing the connections between the primary and secondary wall layers. We also observed that xylogucan is incorporated at a high level in the inner layer of nacreous walls of mature sieve tube elements.

  • 18.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    CELL 12-Chemo-enzymatically modified xyloglucan: A versatile system for biomedical cellulose modification2008Ingår i: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 235Artikel i tidskrift (Övrigt vetenskapligt)
  • 19.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Enzymatic functionalization of cellulosic fibres for textile and other applications: Xyloglucan as a molecular anchor2010Ingår i: Advances in Textile Biotechnology, Elsevier, 2010, s. 266-287Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    The inherent recalcitrance of paracrystalline cellulose, which restricts the performance and applications of cellulosic biofibres, has been circumvented by a biomimetic approach that avoids the limitations inherent in direct chemical methods for cellulose modification. The unique ability of the plant enzyme xyloglucan endo-transglycosylase (XET) to produce chemo-enzymatically functionalized xyloglucan (XG) polysaccharides was harnessed together with the inherently strong interaction of XG with cellulose to surface-anchor the XG derivatives. An overview of the use of XG-XET technology in the functionalization of diverse cellulosic substrates is presented. Specific examples are given to highlight the potential of the system for future applications in textiles, pulp and paper, and biomedicine.

  • 20.
    Brumer, Harry
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Rutland, Mark
    KTH, Skolan för kemivetenskap (CHE), Kemi, Yt- och korrosionsvetenskap.
    Sinnott, M. L.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Zhou, Qi
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Cross-Linking Involving a Polymeric Carbohydrate Material2005Patent (Övrig (populärvetenskap, debatt, mm))
    Abstract [en]

    The present invention relates to a method of cross-linking a polymeric carbohydrate material with a second material by means of a soluble carbohydrate polymer and a crosslinking agent. The present invention furthermore relates to the resulting cross-linked material, to uses of the cross-linked material, as well as to a kit comprising the soluble carbohydrate polymer and the cross-linking agent.

  • 21.
    Brumer, Harry
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Zhou, Qi
    KTH, Tidigare Institutioner, Bioteknologi.
    Baumann, Martin J.
    KTH, Tidigare Institutioner, Bioteknologi.
    Carlsson, Kjell
    KTH, Tidigare Institutioner, Bioteknologi.
    Teeri, Tuula
    KTH, Tidigare Institutioner, Bioteknologi.
    Activation of crystalline cellulose surfaces though the chemoenzymatic modification of xyloglucan2004Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, nr 18, s. 5715-1721Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellulose constitutes an important raw material for many industries. However, the superb load-bearing properties of cellulose are accompanied by poor chemical reactivity. The hydroxyl groups on cellulose surfaces can be reacted but usually not without loss of fiber integrity and strength. Here, we describe a novel chemoenzymatic approach for the efficient incorporation of chemical functionality onto cellulose surfaces. The modification is brought about by using a transglycosylating enzyme, xyloglucan endotranglycosylase, to join chemically modified xyloglucan oligosaccharides to xyloglucan, which has a naturally high affinity to cellulose. Binding of the chemically modified hemicellulose molecules can thus be used to attach a wide variety of chemical moieties without disruption of the individual fiber or fiber matrix.

  • 22. Brun, E.
    et al.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    MacKenzie, L. F.
    Withers, S. G.
    McIntosh, L. P.
    Letter to the Editor: Assignment of selectively C-13-labeled cellopentaose synthesized using an engineered glycosynthase2001Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 21, nr 1, s. 67-68Artikel i tidskrift (Refereegranskat)
  • 23.
    Campuzano, Lain
    et al.
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Brumer, Harry
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Piens, Kathleen
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Sage, Ashley
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Mckenna, Therese
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Langridge, Jim
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Accurate mass analysis of glycoprotein isoforms by electrospray ionisation, orthogonal acceleration time-of-flight mass spectrometry and maximum entropy2004Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, nr 10, s. S130-S130Artikel i tidskrift (Övrigt vetenskapligt)
  • 24. Cartmell, Alan
    et al.
    McKee, Lauren
    KTH, Skolan för bioteknologi (BIO). University of Georgia, United States.
    Pena, Maria J.
    Larsbrink, Johan
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kaneko, Satoshi
    Ichinose, Hitomi
    Lewis, Richard J.
    Vikso-Nielsen, Anders
    Gilbert, Harry J.
    Marles-Wright, Jon
    The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases2011Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed beta-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  • 25. Christiernin, M.
    et al.
    Henriksson, Gunnar
    KTH, Tidigare Institutioner                               , Pappers- och massateknik.
    Lindström, Mikael
    KTH, Tidigare Institutioner                               , Fiber- och polymerteknologi.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Lindström, T.
    Laine, J.
    The effects of xyloglucan on the properties of paper made from bleached kraft pulp2003Ingår i: Nordic Pulp & Paper Research Journal, ISSN 0283-2631, E-ISSN 2000-0669, Vol. 18, nr 2, s. 182-187Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Xyloglucan was adsorbed onto bleached soft-wood kraft pulp followed by preparation and analysis of handsheets with respect to sheet formation as well as sheet mechanical and optical properties. Adsorption of xyloglucan was found to be slow. After more than 20 hrs adsorption, equilibrium had not been reached. The amount of xyloglucan adsorbed increased with beating, but neither the rate of adsorption nor the quantity adsorbed was significantly affected by temperature. Xyloglucan was found to be practically irreversibly adsorbed onto the fibres and the effects of xyloglucan on paper sheet properties were investigated after thorough washing of the pulp. The adsorption characteristics of xyloglucan confirm observations by other authors on other cellulose substrates. Tensile index values for handsheets formed with the xyloglucan-containing pulps were higher than those measured for control pulps with a comparable beating degree. The light scattering coefficient was, however, not affected by xyloglucan adsorption. Hence, the increase in tensile strength is attributed to an increased relative bond strength between the fibres. Tensile strength versus tear strength relationship was similar for pulps with and without xyloglucan, but water retention value and dewatering resistance were lower for the xyloglucan treated pulps than for the reference pulps at the same tensile strength. In addition, formation was improved for pulps with adsorbed xyloglucan. The conclusion is that xyloglucan is a promising wet end additive that decreases the necessity for beating of the pulp and improves the formation of paper.

  • 26. Comfort, Donald A.
    et al.
    Bobrov, Kirill S.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Harris, James M.
    Kulminskaya, Anna A.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kelly, Robert M.
    Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases2007Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 11, s. 3319-3330Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-D-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-D-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-D-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.

  • 27.
    Eklof, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Ruda, Marcus C.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Michael Smith Laboratories, Department of Chemistry, University of British Columbia, Canada.
    Distinguishing xyloglucanase activity in endo-β(1 → 4)glucanases2012Ingår i: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 510, s. 97-120Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ability of beta-glucanases to cleave xyloglucans, a family of highly decorated beta-glucans ubiquitous in plant biomass, has traditionally been overlooked in functional biochemical studies. An emerging body of data indicates, however, that a spectrum of xyloglucan specificity resides in diverse glycoside hydrolases from a range of carbohydrate-active enzyme families including classic "cellulase" families. This chapter outlines a series of enzyme kinetic and product analysis methods to establish degrees of xyloglucan specificity and modes of action of glycosidases emerging from enzyme discovery projects.

  • 28.
    Eklöf, Jens
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    An endo β‐1,4 glucanse, PtEG16‐1 from black cottonwood (Populustrichocarpa) represents an evolutionary link between bacterial lichenases and XTH geneproductsArtikel i tidskrift (Övrigt vetenskapligt)
  • 29.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling2010Ingår i: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 153, nr 2, s. 456-466Artikel i tidskrift (Refereegranskat)
  • 30.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Shojania, S.
    Okon, M.
    McIntosh, L. P.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 22, s. 15786-15799Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The large xyloglucan endotransglycosylase/hydrolase (XTH) gene family continues to be the focus of much attention in studies of plant cell wall morphogenesis due to the unique catalytic functions of the enzymes it encodes. The XTH gene products compose a subfamily of glycoside hydrolase family 16 (GH16), which also comprises a broad range of microbial endoglucanases and endogalactanases, as well as yeast cell wall chitin/β-glucan transglycosylases. Previous whole-family phylogenetic analyses have suggested that the closest relatives to the XTH gene products are the bacterial licheninases (EC 3.2.1.73), which specifically hydrolyze linear mixed linkage β(1→3)/β(1→4)-glucans. In addition to their specificity for the highly branched xyloglucan polysaccharide, XTH gene products are distinguished from the licheninases and other GH16 enzyme subfamilies by significant active site loop alterations and a large C-terminal extension. Given these differences, the molecular evolution of the XTH gene products in GH16 has remained enigmatic. Here, we present the biochemical and structural analysis of a unique, mixed function endoglucanase from black cottonwood (Populus trichocarpa), which reveals a small, newly recognized subfamily of GH16 members intermediate between the bacterial licheninases and plant XTH gene products. We postulate that this clade comprises an important link in the evolution of the large plant XTH gene families from a putative microbial ancestor. As such, this analysis provides new insights into the diversification of GH16 and further unites the apparently disparate members of this important family of proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

  • 31.
    Eklöf, Jens M.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO).
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    The crystal structure of the outer membrane lipoprotein YbhC from Escherichia coli sheds new light on the phylogeny of carbohydrate esterase family 82009Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 76, nr 4, s. 1029-1036Artikel i tidskrift (Refereegranskat)
  • 32. Eneyskaya, E. V.
    et al.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Backinowsky, L. V.
    Ivanen, D. R.
    Kulminskaya, A. A.
    Shabalin, K. A.
    Neustroev, K. N.
    Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp2003Ingår i: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 338, nr 4, s. 313-325Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A beta-D-xylosidase with molecular mass of 250 +/- 5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl P-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1 --> 4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and H-1 and C-13 NMR spectroscopy. The glycosides synthesised, beta-Xyl-1 --> (4-beta-Xyl-1 -->)(n)4-beta-Xyl-OC6H4NO2-p (n = 1 - 5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1 --> 4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2 - 3) while the former liberated PNP from the entire set of substrates synthesised.

  • 33. Eneyskaya, E. V.
    et al.
    Ivanen, D. R.
    Shabalin, K. A.
    Kulminskaya, A. A.
    Backinowsky, L. V.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Neustroev, K. N.
    Chemo-enzymatic synthesis of 4-methylumbelliferyl beta-(1 -> 4)-D-xylooligosides: new substrates for beta-D-xylanase assays2005Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 3, nr 1, s. 146-151Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transglycosylation catalyzed by a beta-D-xylosidase from Aspergillus sp. was used to synthesize a set of 4-methylumbelliferyl (MU) beta-1-->4-D-xylooligosides having the common structure [beta-D-Xyl-(1-->4)](2-5)-beta- D-Xyl-MU. MU xylobioside synthesized chemically by the condensation of protected MU beta-D-xylopyranoside with ethyl 2,3,4-tri-O-acetyl-1-thio-beta-D-xylopyranoside was used as a substrate for transglycosylation with the beta-D- xylosidase from Aspergillus sp. to produce higher MU xylooligosides. The structures of oligosaccharides obtained were established by H-1 and C-13 NMR spectroscopy and electrospray tandem mass spectrometry. MU beta-D-xylooligosides synthesized were tested as fluorogenic substrates for the GH-10 family beta-D-xylanase from Aspergillus orizae and the GH-11 family beta-D- xylanase I from Trichoderma reesei. Both xylanases released the aglycone from MU xylobioside and the corresponding trioside. With substrates having d.p. 4 and 5, the enzymes manifested endolytic activities, splitting off MU, MUX, and MUX2 primarily.

  • 34. Eneyskaya, Elena V.
    et al.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Golubev, Alexander M.
    Ibatullin, Farid M.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kulminskaya, Anna A.
    Transglycosylating and hydrolytic activities of the beta-mannosidase from Trichoderma reesei2009Ingår i: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 91, nr 5, s. 632-638Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1 -> 4) and beta-(1 -> 3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

  • 35.
    Engfeldt, Torun
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Renberg, Björn
    KTH, Skolan för bioteknologi (BIO).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO).
    Nygren, Per-Åke
    KTH, Skolan för bioteknologi (BIO).
    Eriksson Karlström, Amelie
    KTH, Skolan för bioteknologi (BIO).
    Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein2005Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, nr 6, s. 1043-1050Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.

  • 36. Eriksson, T.
    et al.
    Stals, I.
    Collen, A.
    Tjerneld, F.
    Claeyssens, M.
    Stalbrand, H.
    Brumer, Harry
    KTH, Tidigare Institutioner, Bioteknologi.
    Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module2004Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, nr 7, s. 1266-1276Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.

  • 37.
    Ezekiel Mushi, Ngesa
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Biokompositer.
    Nurani, Ghasem
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Utsel, Simon
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Biokompositer.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Zhou, Qi
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Berglund, Lars A.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Biokompositer. KTH, Skolan för kemivetenskap (CHE), Centra, Wallenberg Wood Science Center.
    Soft, bio-inspired chitin/protein nanocomposites: mechanical behavior and interface interactions between recombinant resilin-like proteins and chitin nanofibersManuskript (preprint) (Övrigt vetenskapligt)
  • 38. Fink, H.
    et al.
    Bodin, A.
    Ahrenstedt, Lage
    KTH, Skolan för bioteknologi (BIO), Träbioteknik (stängd 20090101).
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Träbioteknik (stängd 20090101).
    Risberg, B.
    Enhanced endothelial cell attachment on RGD-modified bacterial cellulose2008Ingår i: World Biomater. Congr., 2008Konferensbidrag (Refereegranskat)
    Abstract [en]

    Studies show so far that BC is a promising material for use in the cardiovascular research area. The possibility to easy modify the surface of the BC makes it a good candidate for pre-seeding in vitro with or recruiting of endothelial cell in vivo.

  • 39. Fink, Helen
    et al.
    Ahrenstedt, Lage
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bodin, Aase
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Gatenholm, Paul
    Krettek, Alexandra
    Risberg, Bo
    Bacterial cellulose modified with xyloglucan bearing the adhesion peptide RGD promotes endothelial cell adhesion and metabolism - a promising modification for vascular grafts2011Ingår i: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, Vol. 5, nr 6, s. 454-463Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Today, biomaterials such as polytetrafluorethylene (ePTFE) are used clinically as prosthetic grafts for vascular surgery of large vessels (>5 mm). In small diameter vessels, however, their performance is poor due to early thrombosis. Bacterial-derived cellulose (BC) is a new promising material as a replacement for blood vessels. This material is highly biocompatible in vivo but shows poor cell adhesion. In the native blood vessel, the endothelium creates a smooth non-thrombogenic surface. In order to sustain cell adhesion, BC has to be modified. With a novel xyloglucan (XG) glycoconjugate method, it is possible to introduce the cell adhesion peptide RGD (Arg-Gly-Asp) onto bacterial cellulose. The advantage of the XG-technique is that it is an easy one-step procedure carried out in water and it does not weaken or alter the fiber structure of the hydrogel. In this study, BC was modified with XG and XGRGD to asses primary human vascular endothelial cell adhesion, proliferation, and metabolism as compared with unmodified BC. This XG-RGD-modification significantly increased cell adhesion and the metabolism of seeded primary endothelial cells as compared with unmodified BC whereas the proliferation rate was affected only to some extent. The introduction of an RGD-peptide to the BC surface further resulted in enhanced cell spreading with more pronounced stress fiber formation and mature phenotype. This makes BC together with the XG-method a promising material for synthetic grafts in vascular surgery and cardiovascular research.

  • 40. Gerttula, S.
    et al.
    Zinkgraf, M.
    Muday, G. K.
    Lewis, D. R.
    Ibatullin, Farid M.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. National Research Center Kurchatov Institute, Russian Federation.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. University of British Columbia, Canada.
    Hart, F.
    Mansfield, S. D.
    Filkov, V.
    Groover, A.
    Transcriptional and hormonal regulation of gravitropism of woody stems in populus2015Ingår i: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 27, nr 10, s. 2800-2813Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Angiosperm trees reorient their woody stems by asymmetrically producing a specialized xylem tissue, tension wood, which exerts a strong contractile force resulting in negative gravitropism of the stem. Here, we show, in Populus trees, that initial gravity perception and response occurs in specialized cells through sedimentation of starch-filled amyloplasts and relocalization of the auxin transport protein, PIN3. Gibberellic acid treatment stimulates the rate of tension wood formation and gravibending and enhances tissue-specific expression of an auxin-responsive reporter. Gravibending, maturation of contractile fibers, and gibberellic acid (GA) stimulation of tension wood formation are all sensitive to transcript levels of the Class I KNOX homeodomain transcription factor-encoding gene ARBORKNOX2 (ARK2). We generated genome-wide transcriptomes for trees in which gene expression was perturbed by gravistimulation, GA treatment, and modulation of ARK2 expression. These data were employed in computational analyses to model the transcriptional networks underlying wood formation, including identification and dissection of gene coexpression modules associated with wood phenotypes, GA response, and ARK2 binding to genes within modules. We propose a model for gravitropism in the woody stem in which the peripheral location of PIN3-expressing cells relative to the cambium results in auxin transport toward the cambium in the top of the stem, triggering tension wood formation, while transport away from the cambium in the bottom of the stem triggers opposite wood formation.

  • 41. Ghebremichael, K. A.
    et al.
    Gunaratna, K. R.
    Henriksson, H.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Dalhammar, Gunnel
    KTH, Skolan för bioteknologi (BIO), Miljömikrobiologi.
    A simple purification and activity assay of the coagulant protein from Moringa oleifera seed2005Ingår i: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 39, nr 11, s. 2338-2344Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5 h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1-4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications.

  • 42. Gilbert, Harry J.
    et al.
    Stalbrand, Henrik
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    How the walls come crumbling down: recent structural biochemistry of plant polysaccharide degradation2008Ingår i: Current opinion in plant biology, ISSN 1369-5266, E-ISSN 1879-0356, Vol. 11, nr 3, s. 338-348Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The recent years have witnessed considerable developments in the interpretation of the three-dimensional structures of plant polysaccharide-degrading enzymes in the context of their functional specificity. A plethora of new structures of catalytic, carbohydrate-binding and protein-scaffolding modules involved in (hemi)cellulose catabolism has emerged in harness with sophisticated biochemical analysis. Despite significant advances, a full understanding of the intricacies of substrate recognition and catalysis by these diverse and specialised enzymes remains an important goal, especially if the application potential of these biocatalysts is to be fully realised.

  • 43. Gloster, Tracey M.
    et al.
    Ibatullin, Farid M.
    Macauley, Katherine
    Eklöf, Jens
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Roberts, Shirley
    Turkenburg, Johan P.
    Bjornvad, Mads E.
    Jorgensen, Per Lina
    Danielsen, Steffen
    Johansen, Katja S.
    Borchert, Torben V.
    Wilson, Keith S.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon J.
    Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH122007Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, nr 26, s. 19177-19189Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed hemicellulose. One such hemicellulose is xyloglucan, which displays a beta-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand free and xyloglucan- oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (beta/alpha)(8) and beta-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the beta-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzymeis similarly competent in the degradation of unbranched glucans. In the case of the (beta/alpha)(8) GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

  • 44. Greffe, L.
    et al.
    Bessueille, L.
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides2005Ingår i: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, nr 4, s. 437-445Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel class of nonionic, carbohydrate-based surfactants has been synthesized from the plant polysaccharide xyloglucan. Enzymatic hydrolysis of xyloglucan yielded a series of well-defined, highly branched oligosaccharides that, following reductive amination, were readily conjugated with fatty acids bearing C-8 to C-18 chains under mild conditions. The critical micelle concentration, determined by tensiometry and dye-inclusion measurements, showed a typical dependence on acyl chain length and was sensitive to the degree of galactosylation of the head group. Several compounds from this new group of surfactants, especially those with C-14 and C-16 chains, were useful for the extraction of membrane-bound enzyme markers from different plant cell compartments in catalytically active form.

  • 45.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ibatullin, Farid
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Sundqvist, Gustav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Davies, Gideon
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds2009Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 7, s. 1782-1788Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to Mw 60000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4); L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCel7B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.

  • 46.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Tan, Tien-Chye
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    von Schantz, Laura
    Karlsson, Eva Nordberg
    Ohlin, Mats
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Divne, Christina
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module2010Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 78, nr 3, s. 785-789Artikel i tidskrift (Refereegranskat)
  • 47.
    Gullfot, Fredrika
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    Teeri, Tuula
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Design of GH16 XET/XEH chimeric enzymes with SCHEMA: ManuscriptManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.

    A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.

  • 48. Gunnarsson, Lavinia Cicortas
    et al.
    Zhou, Qi
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Montanier, Cedric
    Karlsson, Eva Nordberg
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Ohlin, Mats
    Engineered xyloglucan specificity in a carbohydrate-binding module2006Ingår i: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 16, nr 12, s. 1171-1180Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

  • 49. Gustavsson, M. T.
    et al.
    Persson, P. V.
    Iversen, T.
    Martinelle, Mats
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase2005Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 6, nr 1, s. 196-203Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M-r xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.

  • 50. Hart, D. O.
    et al.
    He, S. M.
    Chany, C. J.
    Withers, S. G.
    Sims, P. F. G.
    Sinnott, M. L.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase2000Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, nr 32, s. 9826-9836Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase dan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D- lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2,2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2,6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.

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